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Characterization of essential oil profiles, triterpenic acids, and biological assay in aerial parts of various Thymus persicus Jalas (Ronniger ex Rech.f.) populations

Chemical and Biological Technologies in Agriculture volume 10, Article number: 147 (2023) Cite this article

Abstract

Thymus persicus is a valuable natural source of anticancer triterpenic acids (TAs) such as betulinic acid (BA), oleanolic acid (OA), and ursolic acid (UA), which is growing wild in the northwest of Iran. In the present study, variability in morphological characteristics, phytochemical composition, and biological activity among T. persicus populations (TPPs) were investigated. The plants were phenotypically different with the highest variations in some morphological traits. In total, sixty-seven compounds representing 97.2‒99.9% of the essential oils were identified. Thymol (8.1‒43.9%), α-terpineol (1.8‒34.2%), and p-cymene (0.4‒13.4%) were the major components of the studied oils. The content of BA, OA, and UA was ranged as 530.55 ± 13.04‒856.89 ± 6.76, 419.35 ± 11.44‒584.43 ± 12.67, and 941.66 ± 11.49‒1070.82 ± 10.14 mg 100 g‒1 DW in the studied TPPs, respectively. The highest total phenol content (87.26 ± 4.35 mg GAE g–1 DW), total flavonoid content (72.34 ± 2.63 mg QE g–1 DW), and antioxidant property (IC50 = 64.28 ± 4.57 μg ml–1 and 61.68 ± 1.10 μmol Fe+2 g–1 DW) were recorded in TPP1 (Baderlu). The essential oil of the TPP3 (Angooran) showed the lowest minimal inhibitory concentration (MIC) against the bacteria (0.005‒0.080 mg ml–1) and fungi (0.077‒0.100 mg ml–1) among the studied TPPs. Multiple regression analysis showed an associated correlation among morphological, phytochemical characteristics, and biological activities. Canonical correspondence analysis also determined relationship between phytochemical traits and environmental factors. These findings contain valuable data for the conservation and sustainable exploitation of this valuable medicinal plant.

Graphical Abstract

Introduction

Medicinal and aromatic plants (MAPs) are shown to possess a high broad diversity among the widespread worldwide plants. The MAPs have come to the consideration of many countries as a natural chemical wealth [35, 64, 69]. Unluckily, the increased demand for consumption of MAPs puts this valuable asset at risk of degradation. Evidently, about eight percent of the world’s plants (more than 34,000 plant species involved, by considering their medication) are in a hazardous unstable situation that may enhance their erosion and destruction. Thus, a critical focus on the protection and sustainable exploitation of the plant genetic resources is urgently needed [38]. The plants are heterogeneous based on the morphological and phytochemical traits in different climates and natural habitats [63]. Thereby, for the principle and industrial use of MAPs, it is necessary to evaluate the identity and the nature of them from a variety of genetic, morphological, chemical, and manufacturing perspectives [16, 55, 56].

Due to climate diversity, Iran has a vast and distinctive biodiversity, especially for MAPs. Seven thousand five hundred plant species, of which 1700 are MAPs are represented in the Flora of Iran [42]. Of course, any efforts to characterize the morphological and phytochemical diversity of each medicinal plant can lead to the introduction of vulnerable species in the agricultural systems and for the production of new pharmaceutical products as well. The high quality and content of natural products has attracted their application in food and pharmaceutical industries, and their high potential in the agricultural sector as a fungicide, insecticide and herbicide has also been presented [27, 77].

Thymus sp. L. is one of the most important genera of the Lamiaceae family and consists of over 300 species of herbaceous annuals and perennials that are widely distributed throughout the world, especially in the Mediterranean region [59]. Thymus species are known to contain a different class of compounds such as essential oils (EOs), phenolics i.e. tannins [61, 71], saponins [62], and triterpenes [66]. The essential oils and crude extracts of Thymus species are extensively used in the perfumery, food, cosmetic, and pharmaceutical industries [37, 72]. Antiseptic, antioxidative, carminative, bacterial impressionability, antimicrobial, and insecticidal properties of Thymus species have been extensively reported [46, 72].

The genus Thymus is represented in Iran by fourteen species, of which four as T. persicus (Ronniger ex Rech. f.) Jalas, T. daenensis Celak, T. caramanicus Jalas, and T. trautvetteri Klokov & Desj.-Shost are endemic [54]. Thymus persicus, commonly known as "Avishan-e-Irani" is one of the valuable and rare medicinal species which is grown in the restricted region of northwest of Iran. The chemical composition and antibacterial activity of the plant EOs have previously been reported [14, 65]. The aerial parts of T. persicus are also interesting as a source of the three well-known triterpenic acids (TAs) namely, betulinic acid (3β-hydroxy-20(29)-lupaene-28-oic acid, BA), oleanolic acid (3β-hydroxyolean-12-en-28-oic acid, OA), and ursolic acid (3β-hydroxy-12-ursen-28-ic acid, UA) [13, 52]. A wide range of biological activities of TAs including anti-inflammatory and antioxidant, anti-HIV, antifungal, antibacterial, immunomodulatory, antidiabetic, and anticancer activities have been characterized [1, 51]. Various Thymus species have been studied worldwide for their total phenolic content (TPC) and total flavonoid content (TFC). Antioxidant activities of different Thymus species have also been reported [73]. Based on the attractive metabolic profile of T. persicus and likely the high demand for the plant materials, there is an immediate need to protect and exploit this plant in the agricultural system. In vitro propagation, conservation, and cell suspension culture of the plant have been previously reported [11,12,13].

Morphological characterization, as well as the phytochemical assessment, are the main steps in the description and classification of germplasm [9]. Cluster analysis allows analyzing both quantitative and qualitative traits simultaneously and has been employed to assess similarities among genotypes in plant breeding programs. As it could be ascertained, T. persicus populations (TPPs) have not been studied yet. Therefore, the present study aimed to characterize the morphological and phytochemical diversity, and biological activities among TPPs grown in Iran and to detect the connection between the two sets of data by multiple regression analysis. According to the variation of the diversification of T. persicus in morphological and phytochemical traits, as well as antioxidant, antifungal, and antibacterial activities, this study probed the advantages of the characteristics in terms of elucidating of outstanding traits to manipulate in breeding programs, defining as main selection criteria for the high TAs content and desired essential oil chemotype. These findings can be interestingly considered by breeders and farmers for the commercial exploitation.

Materials and methods

Plant materials and chemicals

Aerial parts of the thirty individual plants of T. persicus representing of TPPs were collected from four different localities (Baderlu, Yolgun Aghaj, Angooran, and Gharedash) in the Northwest Provinces of Iran (Table 1, Fig. 1). The individuals were selected from the same age plants. The distance between the sampled individuals and populations in each collection site was at least 100 and 2000 m, respectively. The plant samples were botanically identified by Prof. Ali Sonboli and voucher specimens have been deposited at the Herbarium of Medicinal Plants and Drugs Research Institute (MPH-2232, MPH-2233, MPH-2234, and MPH-2235), Shahid Beheshti University, Tehran, Iran (Table 1).

Table 1 Localities and climatic condition of the studied Thymus persicus populations (TPPs) from Iran
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Fig. 1
figure 1

Geographical distribution of the studied populations of Thymus persicus (A). Wild growing plants in their natural habitats including Baderlu (B), Yolgun Aghaj (C), Angooran (D), and Gharadash (E)

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Standards, reagents, streptomycin, fluconazole, and chemical compounds were supplied from Sigma-Aldrich company (USA). Hydrochloric acid, sulphuric acid, diethyl ether, acetic acid, ethanol, acetone, methanol, dimethylsulfoxide (DMSO), HPLC grade methanol, and phosphoric acid of analytical grade were purchased from Merck Corporation (Germany). Authentic of some essential oil components used as standards for GC-FID analyses were purchased from Merck.

Morphological analysis

Morphological characteristics were measured on thirty samples. Names and morphological traits of each population are listed in Table 2. All the traits except the number of branches, calyx nervure, nodes per shoot, leaves per stem, flowers per inflorescence, inflorescences per plant, seed per inflorescence, and non-numeric morphological characteristics were measured using a ruler and digital caliper.

Table 2 Means comparison of morphological characteristics of the studied Thymus persicus populations (TPPs)
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Essential oil isolation and analysis

The EOs were isolated from the air-dried aerial parts (100 g) of TPPs by hydro-distillation using a Clevenger apparatus recommended by the British Pharmacopeia [22] for 3 h. The content of EOs (mg 100 g –1) was calculated just after isolation, and based on triplicate isolation runs. The isolated oils were then dried over anhydrous sodium sulfate (Na2SO4) and were kept in the freezer (‒20 ºC) until analysis. The oil content (%) was calculated as follows formula [73]:

$${\text{Essential oil content }}\left( \% \right)\, = \,\left[ {{\text{mass of oil obtained }}\left( {\text{g}} \right)/{\text{ mass of dry matter }}\left( {\text{g}} \right)} \right]\, \times \,{1}00.$$

The EOs samples were analyzed by gas chromatography-flame ionization detector (GC-FID) and GC-mass spectrometry (GC–MS), and the chemical constituents were then identified. Analytical conditions were: helium as carrier gas (flow rate, 1.1 ml/min) with ionization voltage of 70 eV, injector temperature 250 ºC, detector temperature 300 ºC, split ratio (1:50), oven temperature program: 60–250 ºC at the rate of 4 ºC/min and then held for 5 min. The analysis was performed on fused silica capillary DB-5 column (30.0 m × 0.25 mm, 0.25 μm). To identify the constituents of the EOs, their mass spectra were compared with those of authentic standards from the internal reference mass spectra library [3]. From the GC data, the retention indices of constituents were calculated against those of n-alkanes (C6 to C24) and the EOs on a DB-5 column under the same chromatographic conditions.

Extraction of triterpene acids and HPLC analysis

Betulinic acid, OA, and UA were extracted from the aerial parts of TPPs following the method reported [11], with some modifications. Dried Powder of plant material (each 1.0 g) was mixed with 40 ml methanol and extracted by sonication (150 W,28 kHz) for 40 min. The obtained methanolic mixture was centrifuged at 10,000 rpm for 10 min. The supernatant was pooled and concentrated in a rotary evaporator at 40 °C (Heidolph Instruments GmbH, Schwabach Germany). The methanolic extract was further separated into organic (30 ml ethyl acetate) and aqueous (30 ml double distilled water) layers. The ethyl acetate phase was collected and evaporated in a rotary evaporator at 40 °C. The dry ethyl acetate extract was dissolved in HPLC grade methanol (10 ml), filtered through a Millipore filter (0.45 mm), and used for analysis. The HPLC instrument properties and proportion of solvents used for TA analysis were according to the method of Bakhtiar et al. [13]. The analysis was performed using HPLC equipped with a 2800 Smartline photo-diode array (PDA) detector with a C18 analytical column (250 × 4.6 mm, 3.5 μm and a UV detector (Waters 2487. The following gradient system was used with methanol-phosphoric acid–water (87:0.05:12.95, v/v/v. The flow was maintained at 0.5 ml/min and column temperature at 25 °C; sample injection was 20 μl. Calibration curves were constructed by injecting separately standard solutions at the seven concentrations of 2, 10, 50, 100, 200, 500, and 1000 ppm. All injections were performed in triplicates. Absorbance was recorded at 210 nm wavelength. System suitability tests were performed by checking the linearity, precision, and recovery of three triterpene acids in the quantification experiment. The calibration curves were prepared by linear regression by a graph informing the area ratio of an external standard.

Determination of total tannins and total saponins content

Total tannin content (TTC) was performed according to Abdouli et al. [2] with some modifications. For instance, powdered dried aerial parts of TPPs (500 mg) were mixed with 5 ml diethyl ether containing 1% acetic acid and were then centrifuged at 2,500 rpm for 5 min. After removing the supernatant, re-extraction was performed with 5 ml of acetone (70%) and shaking for 1 h. The TTC was calculated as the difference in TPC based on the Folin–Ciocalteu method before and after the treatment with polyvinylpyrrolidone (PVP) 4000.

Total saponin content (TSC) of the studied samples was determined as described previously [4] with some modifications. The samples were extracted in a microwave system (Milestone ETHOS UP, Italy). Initially, powdered dried sample (500 mg) was extracted using a microwave-assisted extraction method subjected to irradiation (5 min), 575-Watt microwave power, and 1:10 g ml–1 solid-to-solvent ratio (500 mg sample, 5 ml ethanol). The mixture was then centrifuged at 2000 rpm for 10 min and dried under reduced pressure in a rotary evaporator. After that, 50 μl extract was mixed with 200 ml methanol, 100 μl vanillin-ethanol (10:90 w/v), and 300 μl sulphuric acid (70%) and heated at 100 ͦ C for 5 min. The absorption was read at a wavelength of 540 nm by the spectrophotometer (Bio-Tek Instruments, Inc., USA). The results were expressed as mg diosgenin equivalent per gram dry weight basis (mg DE g–1 DW). Calibration curves (y = 0.0015x + 0.0045, R2 = 0.9999) were plotted using several concentrations of diosgenin (100‒500 mg ml–1). The TSC was determined as follows: [the volume of extraction solvent (ml) × the concentration measured from diosgenin standard curve (mg ml–1]/the dry weight of the sample (g).

Determination of total phenol and total flavonoid content

The total phenolic content (TPC) of the samples was determined by the method of Singleton [70] using gallic acid (GA) as the standard. Briefly, plant extracts (25 μl), Folin–Ciocalteu’s reagent (125 μl), and sodium carbonate (100 μl, 7.0%) were mixed and incubated for 30 min at room temperature. Absorbance was measured at 765 nm against methanol as a blank. Data expressed as mg GA equivalents per g of dry matter (mg GAE g–1 DW). The extraction was conducted in triplicate. The linearity range of the calibration curve was 10 to 1000 μg ml–1 (y = 0.0038x + 0.1579, R2 = 0.9938).

The total flavonoid content (TFC) was determined using the method of Chang et al. [25] with some modifications. In summary, 20 μl solution of the sample, 80 μl of distilled water, 6 μl (0.5 M) sodium nitrite (NaNO2), 6 μl (0.3 M) aluminum chloride hexahydrate (AlCl3.6H2O), and 80 μl (1.0 M) sodium hydroxide (NaOH) were pipetted to plate, respectively. The mixture was allowed to stand for 10 min at room temperature, and absorbance was determined at 510 nm versus the prepared water blank. The average of three readings was used and then expressed as quercetin equivalents (QE) on g dry weight basis (mg QE g–1 DW). The linearity range of the calibration curve was 10 to 1000 μg ml–1 (y = 0.0005x + 0.1270, R2 = 0.9888). The assay for each sample was conducted in triplicate.

Assay of antioxidant properties

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity was determined based on the method of Blois [20] with some modifications. Briefly, 200 μl of methanolic extract (1 mg ml–1) of the plant sample were serially diluted in a 96 well plate and 400 μl of DPPH solution (0.1 mM) was added to each well containing the diluted samples. The negative control was prepared by mixing the DPPH working solution (2 ml) with methanol (1 ml). The solutions were incubated at room temperature for 60 min in the dark. The absorbance values were recorded at 515 nm. DPPH assay was carried out in triplicate for each sample. The inhibition percentage of anti-oxidative activity was determined using the equation: DPPH clearance = Acontrol–Asample)/Acontrol × 100%. The DPPH radical scavenging activity of butylated hydroxytoluene (BHT) was also assayed for comparison. The concentration providing 50% inhibition (IC50) was calculated using a calibration curve in the linear range by plotting the extract concentration vs. the corresponding scavenging effect. IC50 value, representing the amount of extract which scavenged 50% of the DPPH radical, was calculated from percent scavenging versus concentration curve. A higher concentration to reduce 50% of DPPH solution showed lower antioxidant activity. Results were expressed as IC50 μg ml–1.

The ferric reducing-antioxidant assay (FRAP) solutions were prepared as described previously [17]. The reagent was prepared by mixing acetate buffer (20 ml, 300 mmol l–1, pH 3.6), 10 mmol l–1 TPTZ solution (2.5 ml) in 40 mmol l–1 hydrochloric acid and 20 mmol l–1 iron (III) chloride (FeCl3) solution (2.5 ml) in proportions of 10:1:1 (v/v), respectively. The mixture was allowed to react for 30 min at temperature of 37 °C. The absorbance of the mixture was then read at 593 nm. Ascorbic acid was used as the standard curve. The standard curve was constructed using iron (II) sulfate (FeSO4) solution (0.5‒10 mg ml–1). The regression equation was obtained: y = 0.0035x‒0.0030, R2 = 0.9991. The results were expressed as μmol of Fe+2 per gram dry plant weight (μmol Fe+2 g–1 DW).

Absorbance was measured using a spectrophotometer (Bio-Tek Instruments, Inc., USA). All the analyses were run in three replicates and the results were expressed as mean ± standard deviation (SD).

Antimicrobial assay

Four strains of bacteria and fungi were obtained from ATCC (American Type Culture Collection, Rockville, MD, USA). The two gram-positive strains: Staphylococcus aureus (ATCC 33591) and Pseudomonas aeruginosa (ATCC 27853) and two gram-negative strains: Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). The following four fungal strains including Candida albicans (ATCC 90028), C. glabatra (ATCC 90030), C. krusei (ATCC 6258), and C. parapsilosis (ATCC 22019) were used for the antifungal assay.

The bacterial strains were preserved at − 80 °C, subcultured on Mueller–Hinton agar (MHA) medium, and maintained at 4 °C, and grown at 37 °C when required. Stock fungal strains were subcultured on Sabouraud Dextrose Agar (SDA; Merck, Germany) and were maintained at 4 °C until testing was performed. Minimal inhibitory concentration (MIC) was defined as the concentration of test compound at which no macroscopic sign of cellular growth was detected in comparison to the control without compound. Minimal concentrations of bactericidal (MBC) and fungicidal (MFC) were defined as the concentration of compound at which no macroscopic sign of cellular growth was detected compared to the control upon subculturing. The MIC, MBC, and MFC were determined by microdilution method in 96 well microtitre plates described previously [26, 29]. The microbial suspensions of each bacterial and fungal strain were produced from freshly cultured cells in sterile saline that had been adjusted to 1.0 × 105 CFU/well. The EOs were dissolved in 5% DMSO solution that contained 0.10% Tween 80 (v/v) and added appropriate medium with bacterial and fungal inoculum. Essential oils were added in Tryptic Soy Broth (TSB) medium for bacteria, Sabouraud Dextrose Broth (SDB) medium for fungi. The microplates were incubated for 24 h and 48 h at 37 °C for bacteria and fungi, respectively. The MIC of samples was determined following the addition of 40 μl P-Iodonitrotetrazolium violet (INT) 0.2 mg/ml (Sigma I8377) and 30 min of incubation at 37 °C. The lowest concentration with no visible growth was defined as the MBC and MFC, indicating 99.5% killing of the original inoculum. Streptomycin and fluconazole were used as positive controls for bacteria and fungi, respectively. Sterilized distilled water containing 0.1% Tween 80 and 5% DMSO was used as negative control. The experiments were performed in triplicate.

Statistical analysis

Experiments were conducted in triplicates. Analysis of variance for morphological traits, correlation, cluster analysis, and principal components analysis were applied using the SPSS software Version 23. (SPSS Inc., Chicago, IL, USA) and Origin 2021. The significant difference between means were found using Tukey’s multiple range test (p < 0.05) and each of the values was expressed as Mean ± standard error (SE). Pearson product-moment correlation coefficient (r) was used for total characteristics (α = 0.01 and 0.05). The correlation between two sets of data was performed by multiple regression analysis, using a "linear regression analysis" "stepwise" option of SPSS version 23.

Principle component analysis (PCA) was used to illuminate marked changes in the morphological data set. The PCA outcome was used to build biplots to portray the distribution and connection of TPPs concerning GC–MS and HPLC–PDA data. Biplots help with identifying clusters of metabolites that may be associated with the performance or regulations of the plant genotypes. Canonical correspondence analysis (CCA) was assessed using PAST software.

Results

Morphological traits

The morphological characteristics were significantly different between the TPPs (Tables 2 and 3). Among the studied traits, dry matter weight varied ranged from 187.5 (TPP2) and 235.5 g (TPP3). The plant height varied from 7.5 (TPP2) to 9.2 cm (TPP4). The leaf length ranged between 10 (TPP2) and 11.8 cm (TPP4), while leaf width varied from 0.6 (TPP1) to 0.9 cm (TPP4). The number of inflorescences per plant ranged from 128.2 (TPP2) to 159.1 (TPP1). Also, the calyx color varied from green (TPP2) to purple in the rest of the populations. Spearman’s correlation coefficient was positively high if 0.68 < r < 0.97. The results demonstrated a positive and negative correlation (p < 0.01, p < 0.05) between the morphological characteristics. Stem diameter, color, and coat, plant height, bract width, corolla width and coat, seed width, and number of seed per inflorescence had the highest positive and negative correlations with the studied morphological traits (Table 4). Understanding the relationship between the morphological traits helps to select suitable options for breeding programs [32].

Table 3 Codes non-numeric morphological characteristics of the studied Thymus persicus populations (TPPs)
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Table 4 Correlation matrix among the studied characteristics of Thymus persicus populations (TPPs)
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The cluster analysis of morphological data based on UPGMA split populations into two distinct clusters, the first branch was divided into two sub-branches that the first one comprising TPP1 (Baderlu), and the second one included TPP2 (Yolgun Aghaj) and TPP4 (Gharedash). The last completely separated branch was the TPP3 (Angooran) (Fig. 2).

Fig. 2
figure 2

Dendrogram of four Thymus persicus populations based on the morphological characteristic

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Essential oil content and composition

The highest yield of essential oils (w/w%) was recorded in TPP3 (1.2), TPP4 (0.85), TPP2 (0.14), and TPP1 (0.11), respectively (Table 5). In total, 44, 39, 29, and 26 components were identified in TPP1, TPP2, TPP3, and TPP4 representing 97.2, 99.4, 97.2, and 99.9% of the total oils, respectively. Thymol (43.9%), followed by p-cymene (13.4%) and γ-terpinene (11.1%) were the major compounds identified in the TPP3. 4,8-β-epoxy-Caryophyllene (10.7%), α-terpineol (9.5%), and linalool (8.6%) were characterized in the TPP1 as the main essential oil constituents, while α-terpineol (34.2%), thymol (17.7%) and geraniol (10.7%) were the main compounds in the TPP4 oil. The major components of the TPP2 were α-terpineol (23.3%), thymol (13.4%), and geraniol (12.8%) (Table 5). GC–MS chromatograms of the EOs from all TPPs are shown in Fig. 3.

Table 5 Chemical variability in the essential oils of four Thymus persicus populations (TPPs)
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Fig. 3
figure 3

Gas chromatography–mass spectrometry (GC–MS) chromatograms of the essential oil from Baderlu, Yolgun Aghaj, Angooran, and Gharadash populations of Thymus persicus

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The oils were found rich in oxygenated monoterpenes (41.2‒78.8%), followed by monoterpene hydrocarbons (4.7‒35.2%), sesquiterpene hydrocarbons (2.6‒7.3%), and oxygenated sesquiterpenes (0.3‒23.3%). Most of the essential oil samples were rich in terpenes, with the majority of monoterpenes followed by sesquiterpenes and diterpenes (Table 5).

Heatmap analysis classified TPPs into two main groups based on their essential oil compositions (Fig. 4). The group 1 consisted of the TPP2, TPP4, and TPP1 and group 2 included TPP3. The group 1 was further divided into two sub-clusters.

Fig. 4
figure 4

Heatmap of the essential oil profile of the studied Thymus persicus populations. Mean values refer to colors from minimum displayed in bright yellow to maximum represented with dark green

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Variability in phytochemical compounds

The phytochemical traits were significantly different among the studied TPPs (Table 6). Maximum contents (mg 100 g –1 DW) of BA (856.89 ± 6.76), OA (584.43 ± 12.67), and UA (1070.82 ± 10.14) were determined in the aerial parts of TPP1, TPP4 and TPP3, respectively (Table 6). Calibration curves for the standards illustrated good linearity at examined concentrations (2 to 1000 mg l–1), with correlation coefficients (R2) of 0.9991, 0.9994, and 0.9994 for BA, OA, and UA, respectively. Important differences were found in the correlations among the studied TAs. Total tannins content ranged from 246.32 ± 6.87 mg 100 g–1 DW in Angooran (TPP3) to 690.13 ± 9.38 mg 100 g–1 DW in Baderlu (TPP1). The highest TSC (36.78 ± 1.85 mg DE g–1 DW) was observed in Gharadash (TPP4), while the lowest value (18.46 ± 1.22 mg DE g–1 DW) was found in Baderlu (TPP1). The TPC in the extracts of the studied samples ranged from 24.31 ± 1.26 to 87.26 ± 4.35 mg GAE g–1 DW in TPP3 and TPP1, respectively. The highest TFC (mg RE g–1 DW) was found in TPP1 (72.34 ± 2.63), while the lowest content (21.12 ± 1.08) was determined in TPP3 (Table 6).

Table 6 Variability in the content of phytochemical compounds in Thymus persicus populations (TPPs)
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Antioxidant properties

IC50 in TPPs ranged from 209.73 ± 4.32–64.28 ± 4.57 μg ml–1 for TPP3 and TPP1, respectively. The antioxidant power varied from 34.11 ± 1.75–61.68 ± 1.10 μmol Fe+2 g–1 DW. This value in the studied samples was in the order of TPP3 < TPP4 < TPP2 < TPP1.

According to PCA analysis, the studied populations were grouped into four different classes. The first and second PCA for the phytochemical compounds yielded 64.30% and 21.96% of the total variance, respectively (Fig. 5). Along axis 1 of the graph, TPP2 was grouped on the positive region and contributed to carvacrol, α-terpineol, and geraniol. The TPP4 was negatively correlated with TSC, OA, UA, and DPPH. Along axis 2 of the graph, TPP1 formed a separate group on the positive region of the PC2 axis and were associated with TPC, TFC, TTC, FRAP, linalool, β-bisabolene, and 4,8-β-epoxy-caryophyllene. The highest BA, thymol, p-cymene, and γ-terpinene were found in TPP3 that formed a group in the negative section of the PC2 axis (Fig. 5).

Fig. 5
figure 5

Biplot of PCA analysis based on the essential oils composition, HPLC–PDA analysis of triterpenic acids, and other phytochemical compounds

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Antimicrobial activity

The studied EOs of TPPs showed a significant antibacterial activity against gram-positive and gram-negative bacteria (Table 7). The MIC for TPPs was ranged as 0.005‒1.190 mg ml–1, while the MBC was varied from 0.010 to 2.416 mg ml–1. The essential oil of Angooran population (TPP3) had the strongest antibacterial activity. The highest MIC values in TPP3 ranged from 0.005 to 0.080 mg ml–1, and the MBC values varied from 0.010 to 0.160 mg ml–1, depending on the bacteria tested.

Table 7 Antibacterial minimal inhibitory concentration (MIC) (mg ml–1) and minimal bactericidal concentration (MBC) (mg ml–1) of Thymus persicus populations (TPPs)
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Among all tested EOs, generally Angooran population (TPP3) proved to be the most efficacious against all fungi at the lowest concentration applied (MIC 0.077 mg ml–1 and MFC 0.154 mg ml–1 against C. albicans; MIC 0.100 mg ml–1 and MFC 0.201 mg ml–1 against C. glabrata; MIC 0.083 mg ml–1 and MFC 0.167 mg ml–1 against C. krusei; MIC 0.080 mg ml–1 and MFC 0.165 mg ml–1 against C. parapsilosis) (Table 8).

Table 8 Antifungal minimal inhibitory concentration (MIC) (mg ml–1) and minimal fungicidal concentration (MFC) (mg ml–1) of Thymus persicus populations (TPPs)
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The EOs from Baderlu population (TPP1) exhibited a weaker antibacterial (MIC range: 0.780–1.190 mg ml–1; MBC range: 0.156–2.416 mg ml–1) and antifungal (MIC range: 0.250–0.500 mg ml–1; MFC range: 0.500–1.000 mg ml–1) activity against the tested strains. The antimicrobial potential of the EOs tested can be ordered as TPP3 > TPP4 > TPP2 > TPP1. The EOs exhibited different antifungal activities with respect to the geographical region of the plant origin.

Association between phenotypical and phytochemical data

The results of the correlation analysis between the chemical compounds are presented in Fig. 6. The correlation matrix showed the relationships among TTC and TPC (r = 0.95), TFC (r = 0.97), FRAP (r = 0.99), linalool (r = 0.97), thymol (r = –0.98), and β-bisabolene (r = 0.99). A positive correlation of TPC was recognized between the TFC (r = 0.98), FRAP (r = 0.97), and β-bisabolene (r = 0.98). Significant positive correlations between p-cymene and γ-terpinene (r = 1.00), as well as thymol (r = 0.97) were observed. The “r” value for FRAP and linalool (r = 0.99), β-bisabolene (r = 0.99), TPC (r = 0.97), and TFC (r = 0.96) was positive and high, indicating a notable association among these compounds with antioxidant activity in the plant.

Fig. 6
figure 6

Correlation coefficients between phytochemical components on studied Thymus populations

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The correlation (p < 0.01, p < 0.05) between phenotypical and phytochemical data was significant. In particular, factors relating to leaves and flowers, plant height, number of inflorescences per plant, dry matter weight, and number of flowers per inflorescence showed an association with BA, while leaf length and bract length correlated with OA. Similar to BA, UA showed an association with the number of inflorescences per plant and dry matter weight. The TSC had a positive correlation (β = 0.965) with dry matter weight, while had a negative correlation with flower stem length (β = ‒ 0.966). In the EOs, α-terpineol correlated with leaf coat, internode length, and number of flowers per inflorescence. Furthermore, two variables, including the number of flowers per inflorescence and the number of inflorescences per plant showed an association with γ-terpinene. 4,8-β-epoxy-Caryophyllene is associated with the number of inflorescences per plant, root length, and number of flowers per inflorescence (Table 9).

Table 9 Morphological traits associated with phytochemical compositions in Thymus persicus populations (TPPs) as illustrated using multiple regression analysis and coefficients
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Discussion

Phenotypic traits are influenced by genetic factors and environmental conditions, which is very important to investigate these traits as primary studies in introducing plants to breeding and cultivation systems. For this purpose, the diversity of morphological traits has been considered in many medicinal and aromatic plants so far [32, 40]. In this research, morphological and phytochemical traits showed statistically significant variation among the studied populations in each parameter measured. Fattahi et al. [31] obtained similar results about morphological and chemical correlation in the study of Salvia reuterana Boiss. wild populations. Méndez-Tovar et al. [50] found that the morphological characteristics of T. mastichina (L.) L., including the number of flowers per inflorescence, number of inflorescences per plant, bract length, and bract width had the most variation among the studied traits. Morphological changes can be related to the genetic and environmental diversity of the species [39].

Variation in the essential oil content among the plant species collected from different geographical locations has been widely reported. For example, the essential oil content of ten species of Thymus from different geographical regions in Iran was recorded in the range of 0.29% to 3.87% [73]. The essential oil yield of 0.35% has also been reported in Turkish endemic thyme (T. spathulifolius Haussken. & Velen.) [24]. The chemical polymorphism can be due to environmental factors and plant species [52].

Thymol as the major constituent in the EOs of the studied samples including TPP3 (43.9%), TPP4 (17.7%), and TPP2 (13.4%) has also been reported at high content in the other Thymus species [67]. The essential oil of TPP3 contained a high percentage of thymol can be considered as a good source of this valuable compound for further applications. Al-Maqtari et al. [6] have reported the essential oil fraction of T. vulgaris L. rich in oxygenated monoterpenes (56.5% of total oil). This high diversity in the oils has also been reported in the other species [28, 33].

In another study on Thymus species from Ukraine, α-terpineol and carvacrol chemotypes were reported [45]. In the present study, α-terpineol (34.2%) was the dominant compound in the essential oil of TPP4, while carvacrol was ranged from 5.2% to 7.2% among TPPs. Mancini et al. [49] also reported that the variation among the major compounds of Thymus EOs can be due to the biosynthetic relationships between thymol and carvacrol. All these data help us to have a better understanding for future works on this valuable medicinal plant.

Triterpenic acids have been determined in many plant species so far [1, 51, 66]. In the present study, analysis of the same T. persicus extract by HPLC showed the presence of the three major TAs. These compounds had higher contents compared to Origanum vulgare L., Origanum majorana L., Salvia officinalis L., and Melissa officinalis L. [7, 48] although these contents were less than Rosmarinus officinalis L. [8, 19].

Kindil et al. [44] reported the TTC in the aerial parts of six Thymus species from different locations in Croatia in the range of 0.77 ± 0.07% to 1.59 ± 0.04%. In a study on four species of Thymus from Romania, total tannin content was also found in the range of 0.27% to 1.53%, which was higher than the values obtained in TPPs in the present study. However, the TTC in T. vulgaris (0.27‒0.94%) was near to TPPs [23]. In another study, tannins content (mg catechin g–1) in leaves and stems of four local Moroccan species Thymus was found in the range of 1.7 ± 0.049 to 22.6 ± 0.512. The phytochemical screening of their plant materials revealed an abundance of tannins and flavonoids and the absence of saponins in the stems and leaves of some species [68]. It proposed that the difference in the SMs of different MAPs can be related to genetic, ontogenic, morphogenetic, and environmental factors [75].

All studied samples exhibit high TPC. Variations in TPC and antioxidant activity (0.8‒48,680 μg ml–1) have been reported for Thymus species [72]. In a study on three Thymus species, high TPC and TFC were detected in T. kotschyanus Boiss. & Hohen. (337.0 ± 8.31 mg rutin mg–1) and T. pubescens Boiss. & Kotschy ex Celak. (50.39 ± 0.75 mg rutin mg–1), respectively. The highest antioxidant activity was also reported for T. pubescens (IC50 = 31.47 μg ml–1) [58]. Thymus species are the best sources of chemical components and antioxidant agents for the cure of many diseases. The extracts from TPPs showed high value of TPC, TFC, and antioxidant activity, so these extracts can be used as antibiotics or preservatives in the pharmaceutical and food industries.

Essential oils are known to have inhibitory activity against a variety of microbes [41]. The EOs of the Lamiaceae members have been shown strong antimicrobial activity [60, 76]. Origanum vulgare and T. vulgaris are the most studied EOs exhibiting antimicrobial activity against a wide range of bacterial and fungi strains [21]. The antimicrobial activity of the EOs of Thymus species is well documented in the literature for T. vulgaris [5], T. daenensis L. [53], T. zygis L. and T. mastichina [15], T. maroccanus Ball and T. broussonetii Boiss. [30], and T. caramanicus Jalas [57] so far. It has been reported that EOs of the plant with MIC of 2 mg ml–1 or lower show significant antimicrobial activity [34, 74]. Therefore, the EOs of the TPPs could be considered as a potent and valuable antimicrobial agent for further exploitation in food and pharmaceutical products.

The results are similar to those of Khadivi-Khub et al. [43] on Satureja mutica Fisch. & C.A. Mey. Also, carvacrol showed associations with flower stem length and dry matter weight. Based on the results of multiple regression analysis, leaf and flower variables were associated with phytochemical compounds, which showed the main role of these morphological traits in the production of these compounds. This finding was in agreement with the obtained results by Berardi et al. [18]. Studies on correlations between morphological and phytochemical traits can help plant breeder’s select suitable populations.

The studied TPPs are distributed within the latitude of 36° 26´ N to 36° 45´ N and longitude of 47° 13´ E to 47° 26´ E encompassing different geographical regions. All populations were located in the northwest of Iran, and their mean rainfall is between 340 and 390 mm/year. To evaluate the correlation between environmental factors and the essential oil components, canonical correspondence analysis (CCA) was performed based on the three environmental factors and five important main components of the plants EOs, including thymol, α-terpineol, 4,8-β-epoxy-caryophyllene, p-cymene, and γ-terpinene (Fig. 7). Involved environmental factors were mean annual precipitation (MAP), altitude, and mean annual temperature (MAT). The first CCA variable (CC1) concerning environmental parameters showed that MAP and altitude had a positive share, while MAT had a negative share on this CCA construction. Also, this analysis highlights the role of each environmental factor in the grouping of TPPs. By considering these data, the first canonical variable in connection to the phytochemical characteristics showed that the thymol, p-cymene, and γ-terpinene had a negative share in the formation of CCA1 variables.

Fig. 7
figure 7

Canonical correspondence analysis biplot of Thymus persicus populations, linking percentages of the major and important constituents, collected from different environmental conditions

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4,8-β-epoxy-Caryophyllene and α-terpineol had a positive share with altitude and MAP. Also, thymol correlated with p-cymene and γ-terpinene, and is distinct from α-terpineol and E-caryophyllene. The most important factor of the second CCA (CCA2) was MAT. The three groups were identified based on the PCA and the cluster analyses. According to the analysis of UPGMA (heatmap), the TPP3 collected from the northwest region of Iran is characterized by a high content of thymol. This population was collected from a location with high temperature, low rainfall, and relatively low elevation. In general, it may be assumed that the content of essential oil and thymol is high in arid and semi-arid conditions [47], as illustrated in this CCA analysis. The correlation of thymol with p-cymene and γ-terpinene was not only obvious but also distinct from them. The distance might be due to the biosynthesize pathway. The main precursors for the biosynthesis of thymol are γ-terpinene and p-cymene [10]. The higher content of γ-terpinene and p-cymene to produce thymol as a finished product can lead to a decrease in the content of precursors. Furthermore, the heatmap cluster placed γ-terpinene and p-cymene together, which showed their correlation. The present study investigated the role of some environmental factors. However, phytochemicals can also be attributed to genetics [36].

Conclusions

In this research, morphological and phytochemical characteristics and biological activities of TPPs were evaluated for the first time. Morphological analysis of TPPs showed a high diversity between qualitative and quantitative traits that help the breeder to select the desired genotype. Analysis of the EOs exhibited high diversity among major compounds. Thymol was the most abundant one that present in TPP3. Other major constituents were α-terpineol, p-cymene, geraniol, γ-terpinene, and (E)-caryophyllene. Assessment of the extracts represented considerable contents of anticancer compounds (BA, OA, and UA), TTC, TSC, TPC, TFC, and antioxidant and antimicrobial activity, which can be utilized in scaling up through biotechnological methods. Association and the relationship between various characters are good tools to select the best plant for future breeding programs. It also helps to distinguish of correlation between chemical, morphological, and environmental characteristics. The results showed that extracts and EOs of TPPs can be exploited in food and pharmaceutical industries.

Data availability

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

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Acknowledgements

The authors express their appreciation to the Research Council of Shahid Beheshti University, Tehran, Iran for their support. We also wish to thank Prof. Ali Sonboli and Mr. Hamid Ahadi for their kind help in plant materials collection and HPLC analysis, respectively.

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Authors and Affiliations

  1. Department of Agriculture, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, 1983969411, Iran

    Ziba Bakhtiar, Mohammad Hossein Mirjalili & Mohammad Selseleh

  2. Department of Horticulture, College of Agriculture & Natural Resources, Hormozgan University, Bandar Abbas, Iran

    Alireza Yavari

  3. Department of Medicinal Plants, Faculty of Agriculture and Natural Resources, Arak University, Arak, 38156-8-8349, Iran

    Mansour Ghorbanpour

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  1. Ziba Bakhtiar
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  2. Mohammad Hossein Mirjalili
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  3. Mohammad Selseleh
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  5. Mansour Ghorbanpour
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Contributions

ZB: Conceptualization, Investigation, Data interpretation, Formal analysis, Writing-Original draft. MHM: Supervision, Methodology, Funding acquisition, Data curation, Validation, Writing, Review & editing. MS: Review & editing, Data interpretation, Formal analysis, Visualization. AY: Resources, Review & editing. MG: Data curation, Formal analysis, Validation, Review & editing.

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Correspondence to Mohammad Hossein Mirjalili or Mansour Ghorbanpour.

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Bakhtiar, Z., Mirjalili, M.H., Selseleh, M. et al. Characterization of essential oil profiles, triterpenic acids, and biological assay in aerial parts of various Thymus persicus Jalas (Ronniger ex Rech.f.) populations. Chem. Biol. Technol. Agric. 10, 147 (2023). https://doi.org/10.1186/s40538-023-00520-6

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